Abstract
Purpose :
Retinal degeneration protein 3 (RD3) is crucial for photoreceptor cell survival and has been linked to Leber Congenital Amaurosis type 12 (LCA12), a hereditary retinal disease in humans. RD3 inhibits retinal guanylyl cyclase RetGC1 (human GUCY2D) in vitro by binding to the cyclase. RD3 competition with guanylyl cyclase activating proteins (GCAPs) in photoreceptor inner segments prevents fast degeneration of rods and cones. RD3 is also required for normal accumulation of the cyclase in rod and cone outer segments. The aim of the study was to identify the residues on the RD3 surface that create the interface for its inhibitory binding to RetGC1.
Methods :
Surface-exposed amino acid residues in human RD3 were selected for mutagenesis based on the recently determined 3D NMR structure of RD3 [1]. Mutations were introduced using conventional "splicing by overlap extension” technique. The RD3 mutants were expressed in E. coli, purified, and tested in RetGC1 activity inhibition assay in vitro [2].
Results :
Surface-exposed amino acids were altered using single-point mutations and a few C-terminal deletions. In addition to previously reported internal residues that affected interactions between helices forming the backbone of the RD3 interface for the cyclase [1], we identified several side chains on the surface that were most critical for the ability of RD3 to inhibit RetGC1-GCAP1 complex. The surface-exposed residues essential for cyclase inhibition were clustered in two regions located on the opposite sides of the RD3 molecule. One region included the central part of the α-helical bundle, previously shown to be important for the interaction between RD3 and RetGC1 [1,2]. Substitutions of several solvent-exposed side chains in the helix 3 of the molecule each caused particularly significant, >10 fold, reduction in the RD3 affinity for RetGC1. Another group of amino acid residues critical for the high-affinity binding of RD3 to RetGC1 was located in the loop between helices 1 and 2.
References: [1] Peshenko et al. (2019) J. Biol. Chem. 294, 2318-28. [2] Peshenko et al. (2016) J. Biol. Chem. 291, 19713-23.
Conclusions :
In the present study, we identified two regions on the surface of RD3 that were essential for the regulatory contact with RetGC1.
This is a 2020 ARVO Annual Meeting abstract.